Jef F. G. M. Wintermans

Spectrophotometric characteristics of chlorophylls a and b and their phenophytins in ethanol

Abstract The absorption spectra of chlorophylls a and b in 96% ethanol, and of pheophytins a and b in 80% ethanol-0.01 N hydrochloric acid have been determined in comparison with known absorption coefficients.

Galactolipid transformations and photochemical activities of spinach chloroplasts

Abstract 1. When spinach chloroplasts are isolated in media containing high concentrations of NaCl, monogalactolipase activity can be observed. In media containing sucrose or mannitol, there is also an enzymatic transacylation which results in a decrease in digalactolipid and the formation of acylated derivatives of the mono- and digalactolipids. The rate of release of free fatty acids is slower under the latter conditions. 2. In order to establish a relation between these phenomena and the photochemical capacities of the chloroplasts, several light reactions have been measured in fresh and aged chloroplasts. 3. Hill reactions in Photosystem II and NADP + reduction in Photosystem I are not directly influenced by the lipid transformations. 4. Photophosphorylation and Hill reaction at limiting light intensities are very labile under the experimental conditions. Lipid transformations may contribute to this effect. 5. Serum albumin has no effect on the transformations of chloroplast galactolipids. It has a stimulating and stabilizing effect on photophosphorylation and improves the efficiency of photophosphorylation. Only a part of this effect can be explained by the known capacity of serum albumin to bind free fatty acids.

Localization of galactolipid: galactolipid galactosyltransferase and acyltransferase in outer envelope membrane of spinach chloroplasts

Abstract We have measured the localization within spinach chloroplast envelope membranes of two galactolipidmanipulating enzymes, the galactolipid:galactolipid galactosyltransferase and the galactolipid:galactolipid acyltransferase. Both are localized on the outer envelope membrane. This situation differs strikingly from the localization of the enzymes involved in monogalactosyldiacylglycerol synthesis, on the inner envelope membrane.

Characterization of galactosyltransferases in spinach chloroplast envelopes

Abstract Two galactosyltransferases involved in the galactolipid metabolism of spinach chloroplast envelopes were studied by specific assays: UDPgalactose: 1,2-diacylglycerol galactosyltransferase, which synthetizes monogalactosyldiacylglycerol from UDPgalactose plus diacylglycerol; and galactolipid: galactolipid galactosyltransferase (GGGT), which forms di- , tri- and tetragalactosyldiacylglycerol by dismutation of galactosyl groups between two galactosyldiacylglycerols. In the assay developed for UDPgalactose: 1,2-diacylglycerol galactosyltransferase, chloroplast envelope membranes were mixed with liposomes of phosphatidylcholine, and the mixture was treated with phospholipase C (from Bacillus cereus). The diacylglycerol produced from phosphatidylcholine is used by UDPgalactose: 1,2-diacylglycerol galactosyltransferase for synthesis of monogalactosyldiacylglycerol in the presence of UDPgalactose. Several characteristics of this enzyme were studied, including the effect of substrate concentrations, pH and temperature. UDPgalactose: 1,2-di-acylglycerol galactosyltransferase did not require cations for activity, but was stimulated by Mg2+ and Mn2+. Of the mono- and divalent cations tested only Zn2+, Cd2+ and Fe2+ were inhibitory. Specific inhibitors for UDPgalactose: 1,2-diacylglycerol galactosyltransferase were UDP and N-ethylmaleimide. In contrast to UDPgalactose: 1,2-diacylglycerol galactosyltransferase, galactolipid: galactolipid galactosyltransferase was strongly stimulated by a series of mono- and divalent cations, most stimulatory being Mn2+ Ba2+, Ca2+ and Mg2+. Galactolipid: galactolipid galactosyltransferase was specifically inhibited by low concentrations of Zn2+ (1 mM) and by chelating anions. UDPgalactose: 1,2-diacylglycerol galactosyltransferase synthetized monogalactosyldiacylglycerol from various molecular species of diacylglycerol; the highest activity was measured with distearoylglycerol and dioleoylglycerol. On the other hand, digalactosyldiacylglycerol synthesis by galactolipid: galactolipid galactosyltransferase proceeded most rapidly by galactosyl transfer to hexaene species of monogalactosyldiacylglycerol.

Separation of chloroplast polar lipids and measurement of galactolipid metabolism by high-performance liquid chromatography

Procedures are described for the separation of polar lipids from plant chloroplasts by high-performance liquid chromatography, using a polar-modified silica column. Glycolipids and phospholipids were eluted with a gradient of 2-propanol/n-hexane (80:55, v/v) and 2-propanol/n-hexane/water/methanol (80:55:15:10, v/v). The lipids were detected by uv absorbance at 202 nm. Diacylglycerol and mono-, di-, and trigalactosyldiacylglycerol and phosphatidylcholine were separated on a LiChrosorb NH2 column (7-microns particles, Merck, FRG), but acidic lipids were retained. These lipids could be quantified from their 202-nm absorbance recording. The absorption coefficients obtained depended on the mean number of double bonds in the different lipid classes. The separation was applied for a rapid monitoring of the lipid composition in thylakoids and in fractionated inner and outer envelopes. The activities of galactosyltransferases involved in galactolipid metabolism, UDPGal:diacylglycerol galactosyltransferase and galactolipid:galactolipid galactosyltransferase, could be measured quantitatively in specific assays for both enzymes.

Spinach chloroplasts: localization of enzymes involved in galactolipid metabolism

Abstract A shortened procedure for the separation of spinach chloroplast envelope membranes is presented, resulting in high yields of enriched inner and outer envelopes. Fractionated envelopes were tested for activities in galactolipid synthesis by measuring incorporation of UDPGal and by using a specific assay for galactolipid: galactolipid galactosyltransferase (GGGT); additionally, acyl-CoA synthetase activities were tested. UDPGal incorporation and GGGT were found predominantly in enriched inner envelopes, acyl-CoA synthetase was predominantly in enriched outer envelopes. These observations point to a localization of both UPDGal: diacylglycerol galactosyltransferase (UDGT) and GGGT in the inner membrane. Alternatively, chloroplasts were incubated with [14C]acetate under conditions permitting fatty acid synthesis in the presence and absence of UDP[3H]Gal. After incubation, envelope fractions were isolated and analyzed for labeled lipids. Analysis agreed with localization of UDGT in the inner envelope. However, labeled products of GGGT were found both in enriched inner and outer envelopes. Furthermore, inactivation of GGGT by the proteinase thermolysin indicates a location on the cytosolic face of the chloroplast. These apparently contradictory results concerning GGGT might be explained by the involvement of contact sites between inner and outer envelopes or by lipid transformation during membrane fractionation.

On the Synthesis of Digalactosyldiacylglycerol in Chloroplasts, and Its Relation to Monogalactolipid Synthesis

About a quarter century after the first observation that galactolipids are specific membrane lipids of chloroplasts (1), it seems appropriate to make a short survey of the knowledge gained about their biosynthesis. In contrast to other papers (2–5) we shall emphasize on the synthesis of digalactosyldiacylglycerol (DGDG*), which has been a subject of much confusion.

Characterization of Galactosyltransferases in Spinach Chloroplast Envelope Membranes Applications of an Assay for UDPGal: Diacylglycerol Galactosyltransferase

Two enzymes involved in the galactolipid metabolism of spinach chloroplast envelope membranes were studied. UDPGal: diacylglycerol galactosyltransferase (UDGT) synthetizes monogalactosyldiacylglycerol (MGDG) from UDPGal plus diacylglycerol (1, 2):

Characterization of Galactosyltransferases in Spinach Chloroplast Envelope Membranes. UDPgal-Dependent and -Independent Galactolipid Synthesis

{\text{UDPGal + diacylglycerol}} \to {\text{UDP + MGDG}}